ABSTRACT
OBJECTIVE Toprepareapolyclonalantibodyforspindlin1protein,anovelcancer related protein,and to provide the data for a better understanding of its functions and screening tu mor. METHODS Purifiedspindlin1proteinwasinjectedintorabbitstoproducethepolyclonalantiserumafter removing glutathione S-transferase (GST)from the fusion protein spindlin 1-GST that was expressed in Escherichia coli..The antiserum was purified through the Hitrap Protein A system,and the titer of spin-dlin 1 polyclonal antibody was detected by ELISA.The specificity of the polyclonal antibody was deter-minedbyWesternblottingandimmunohistochemistry.RESULTS Thetiterofspindlin1polyclonalanti-body was 1∶2000.Western blotting detection demonstrated that the spindlin 1 polyclonal antibody recog-nized myc-spindlin 1 reco mbinant fusion protein in HeLa cells transfected with pAdeasy-myc-spindlin 1 , which also corresponded with Myc.antibody.The HeLa cells were transfected with enhanced green fluo-rescence protein (EGFP)and spindlin 1 vector(pEGFP-C3-spindlin 1 ),which was confirmed by the in-dependent GFP fluorescence assay.The results of immunohistochemistry detection with the spindlin 1 polyclonal antibody suggested that spindlin 1 was mainly expressed in the nuclei of HeLa cells.More i m-portantly,in i mmunohistoche mical assays,the spindlin 1 antibody recognized nuclear spindlin 1 expres-sioninclinicalovariancancertissues.CONCLUSION Thespecificspindlin1polyclonalantibodyispre-pared,which may be used to detect cancer-related protein spindlin 1 in HeLa cells and ovarian cancer tissues.
ABSTRACT
The transcriptional repressor RE1 silencer transcription factor(NRSF/REST) is an important factor that restricts some neuronal traits in neurons.Since these traits are also present in pancreatic islet cells,NRSF-regulated genes involved in islet function are searched.A NRSE-like motif was analysed in human insulin promoter.The role of NRSE was evaluated by generating a model of insulin-secreting cells that firmly express NRSF.The presence of NRSF led to a decrease in activity of human insulin promoter by stable or transient transfection with human insulin-promoter luciferase.The predicted NRSE-like motif also confers NRSF-dependent transcriptional repression in the context of a surrogate gene promoter.Specific binding activity of NRSF/REST to the NRSE-like motif was confirmed by EMSA.Moreover,the binding activity is competed by consensus NRSE sequence.These data showed that human insulin promoter is regulated by the transcriptional repressor NRSF/REST via the NRSE-like motif.